A variety of techniques are currently available for efficient amplification of nucleic acids even from a few starting nucleic acid templates resulting in a large number of amplified products. These include polymerase chain reaction (PCR), rolling circle amplification (RCA) and strand displacement amplification (SDA). Due to higher amplification efficiencies of these techniques, even the slightest contamination of the reagents/reagent solutions employed in such amplification reactions with an undesired nucleic acid molecule may result in a huge amount of false amplification products. If such an amplification were used for diagnostic applications, this would likely result in a false-positive diagnosis.
Reagents or reagent solutions that are used in nucleic acid amplification reactions may get contaminated in various ways. For example, contamination may arise from carry-over amplification product (amplicons) of previous amplification reactions, from the site from which the sample for amplification is collected, by exogenous DNA in the laboratory environment or from reagents or reagent solutions used for amplification reaction.
Various pre-amplification sterilization procedures have been developed to minimize amplicon carry-over. For example, deoxythymidine triphosphate (dTTP) is substituted for deoxyuridine triphosphate (dUTP) in PCR amplifications to make PCR products distinguishable from template DNA. Use of enzyme uracil-N-glycosylase (UNG) in a pre-amplification step cleaves the carry-over amplicons at the incorporated uracil residues. In amplification reactions using the same primers and the same target sequences, enzymatic removal of amplicons from previous similar amplification reactions has also been reported. These methods take advantage of the fact that the contaminant amplicon carries its primer sites at or near the ends of the molecule whereas virtually all other template DNA molecules not arising themselves from a previous PCR reaction, do not have their primer sites so located.
Single strand-specific exonuclease has been used for amplicon de-contamination during strand displacement amplification (SDA) reaction wherein either (or both) the target nucleic acid or the amplicons are in single stranded form. In such methods, even though both the target and amplicons are attacked, due to the short length of amplicons (25-2,000 nucleotides) and their lack of secondary structures, the amplicons are preferentially cleaved.
Use of selectively activable enzymes such as micrococcal nuclease and of DNA-binding agents have been employed to de-contaminate the reagent solution. Enzymatic, physical or chemical pre-treatment of the sample has also been employed to remove or inactivate a contaminating DNA that is originating from the site from where the sample is collected.
Apart from amplicon carry-over, reagents and reagent solutions commonly used to amplify nucleic acids may contain unwanted nucleic acid contaminants that could potentially interfere with standard nucleic acid amplification protocols and procedures. Contaminating DNA may be much longer than that of a primer or an amplicon and specific information about the contaminating DNA may often be minimal. During amplification reactions, false amplification products may also be formed by the inherent contamination of the reagents used for such reactions. For example, polymerization enzymes such as DNA polymerases that are used in amplification reactions may inherently carry contaminating nucleic acids. Standard protein purification techniques might not be sufficient enough to de-contaminate such nucleic acid-binding proteins effectively. So, there exists a need for specific treatments to de-contaminate the reagents and the reagent solutions used for amplification reactions.